The impact of mass spectrometry in life science has been increasingly draw attentions in the last two decades. As interests to mass spec applications getting broader and the techniques penetrate into each research area getting deeper, more challenges have been created. One of the challenges from proteomic researches is dealing with more complex samples with higher throughputs and wider dynamic range. Existing instruments and technologies are still far from meeting researchers demand. The goal of the proposed work is to develop a novel mass spectrometer and the associate methods which could provide much better peptide coverage at higher throughput for complex protein samples with lower detection limits, especially for low abundant proteins. This project will focus on tandem two-dimensional mass spectrometer and its applications. The new instrument will obtain complete MS/MS information, include fragmentation ions from all precursors with their parent-daughter linkage. The ions from the ion source will not be filtered out or under discriminatory selection, instead each of them will be fragmented to get MS/MS spectra, resulting much higher ion usage efficiency. Under phase I investigation, a prototype hybrid Qit-q-TOF instrument was built for 2D MS/MS experiments. Interfaces between each components have been studied, especially for ion trap ion ejection. Results from both computer simulation and experiments demonstrated the kinetic energy of ejected ions are low enough for interfacing TOF analyzer by using proposed reverse scan resonance ejection, which further approved Qit-q-TOF concept is technically feasible for 2D MS/MS. Under phase II funding, we will continue to improve the performance, eventually achieve the requirements of a commercial instrument. With the collaboration to cutting edge research groups, key applications and informatics tools will be developed.